© 2017. Published by The Company of Biologists Ltd | Biology Open (2017) 6, 439-448 doi:10.1242/bio.022467 RESEARCH ARTICLE tBRD-1 and tBRD-2 regulate expression of genes necessary for spermatid differentiation Ina Theofel1, Marek Bartkuhn2, Thomas Boettger3, Stefanie M. K. Gärtner1, Judith Kreher4, Alexander Brehm4 and Christina Rathke1,* ABSTRACT 1998). Transcription of the corresponding genes (spermiogenesis- Male germ cell differentiation proceeds to a large extent in the relevant genes) depends on two testis-specific transcription absence of active gene transcription. In Drosophila, hundreds of complexes: the testis meiotic arrest complex (tMAC), and the genes whose proteins are required during post-meiotic spermatid testis-specific TFIID complex, which consists of testis-specific differentiation (spermiogenesis) are transcribed in primary TATA box binding protein-associated factors (tTAFs) (Beall et al., spermatocytes. Transcription of these genes depends on the 2007; Hiller et al., 2004, 2001). Recruitment of tTAFs to chromatin sequential action of the testis meiotic arrest complex (tMAC), requires the coactivator complex Mediator, and localization of Mediator complex, and testis-specific TFIID (tTFIID) complex. How Mediator subunits to chromatin depends on tMAC (Lu and Fuller, the action of these protein complexes is coordinated and which 2015). Based on these data, it has been suggested that Mediator acts other factors are involved in the regulation of transcription in as a key factor in a tTAF- and tMAC-dependent gene regulatory spermatocytes is not well understood. Here, we show that the cascade that leads to transcriptional activation of spermiogenesis- bromodomain proteins tBRD-1 and tBRD-2 regulate gene relevant genes (Lu and Fuller, 2015). expression in primary spermatocytes and share a subset of Acetylated lysines of histone play an important role in gene target genes. The function of tBRD-1 was essential for the sub- transcription (Sanchez and Zhou, 2009). These histone cellular localization of endogenous tBRD-2 but dispensable for its modifications are recognized by bromodomain-containing protein stability. Our comparison of different microarray data sets proteins (Dhalluin et al., 1999). The bromodomain forms a well- showed that in primary spermatocytes, the expression of a defined conserved structure within functionally distinct proteins, such as number of genes depends on the function of the bromodomain histone acetyltransferases, chromatin-remodeling factors, proteins tBRD-1 and tBRD-2, the tMAC component Aly, the transcriptional co-activators and mediators, and members of the Mediator component Med22, and the tTAF Sa. bromodomain and extra-terminal (BET) family (Josling et al., 2012). Members of the BET family are characterized by having one KEY WORDS: Testis-specific transcription, tTAFs, tMAC, Mediator (in plants) or two (in animals) N-terminal bromodomains and a complex, BET proteins conserved extra-terminal domain that is necessary for protein– protein interactions (Florence and Faller, 2001; Matangkasombut INTRODUCTION et al., 2000; Platt et al., 1999). BET proteins contribute to In Drosophila melanogaster and mammals, the post-meiotic transcription mainly by recruiting protein complexes, e.g. phase of spermatogenesis (spermiogenesis) is characterized by transcription factors and chromatin remodelers (Josling et al., extensive morphological cell changes (Rathke et al., 2014). In 2012; Krogan et al., 2003; Matangkasombut et al., 2000). In flies, transcription almost ceases as the cells enter meiotic mammals, the BET proteins BRD2, BRD3, BRD4, and BRDT are division; therefore, these changes mainly rely on proteins arising expressed in male germ cells (Klaus et al., 2016; Shang et al., 2004). from translationally repressed and stored mRNAs synthesized in BRDT is involved in gene expression during spermatogenesis, primary spermatocytes (Olivieri and Olivieri, 1965; White- among other roles (Berkovits et al., 2012; Gaucher et al., 2012), but Cooper et al., 1998). Hence, a tightly regulated gene transcription the functions of BRD2, BRD3, and BRD4 in male germ cells are not program is required to ensure proper spermiogenesis and male well understood. fertility. In Drosophila, three testis-specific bromodomain proteins In primary spermatocytes, numerous transcripts are synthesized (tBRDs) have been described (Leser et al., 2012; Theofel et al., and translationally repressed (Fuller, 1993; White-Cooper et al., 2014). tBRD-1 contains two bromodomains, is essential for male fertility, and partially co-localizes with tTAFs in primary 1Philipps-Universität Marburg, Department of Biology, Marburg 35043, Germany. spermatocytes (Leser et al., 2012). Likewise, the BET family 2Institute for Genetics, Justus-Liebig-Universität, Giessen 35392, Germany. 3Department of Cardiac Development and Remodeling, Max Planck Institute for members tBRD-2 and tBRD-3 partially co-localize with tBRD-1 Heart and Lung Research, Bad Nauheim 61231, Germany. 4Philipps-Universität and tTAFs in primary spermatocytes (Theofel et al., 2014). In Marburg, Institute of Molecular Biology and Tumor Research, Marburg 35037, addition, subcellular localization of the three tBRDs depends on Germany. both tTAF function and the level of acetylation within the cell (Leser *Author for correspondence ([email protected]) et al., 2012; Theofel et al., 2014). Loss of tBRD-1 function leads to an altered distribution of tBRD-2 and tBRD-3 and to a significant C.R., 0000-0001-9320-8510 down-regulation of a subset of tTAF target genes (Theofel et al., This is an Open Access article distributed under the terms of the Creative Commons Attribution 2014). Protein–protein interaction studies have revealed that tBRD- License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 forms homodimers and also heterodimers with tBRD-2, tBRD-3, and tTAFs (Theofel et al., 2014). The loss of tBRD-1 or tBRD-2 Received 17 October 2016; Accepted 23 February 2017 leads to similar post-meiotic phenotypes, e.g. nuclear elongation Biology Open 439 RESEARCH ARTICLE Biology Open (2017) 6, 439-448 doi:10.1242/bio.022467 defects (Kimura and Loppin, 2015; Leser et al., 2012). It has been (Fig. S1B). We then analyzed the localization of endogenous postulated that in primary spermatocytes, tBRDs cooperate with tBRD-2 in heterozygous and homozygous tbrd-1 mutants and in tTAFs to regulate expression of selected spermiogenesis-relevant heterozygous and homozygous tbrd-1 mutants expressing a genes (Theofel et al., 2014). tBRD-1-eGFP fusion protein (Fig. 1). Western blot analyses Here, we show that a tbrd-1-eGFP transgene restores not revealed that endogenous tBRD-2 levels were not reduced in only male fertility of tbrd-1 mutants but also localization of tbrd-1 mutant testes (Fig. 1A). In heterozygous tbrd-1 mutant tBRD-2 to chromosomal regions. Protein–protein interaction spermatocyte nuclei, endogenous tBRD-2 localized to studies demonstrated that both bromodomains are dispensable for chromosomal regions, nucleolus, and nuclear speckles in the tBRD-1 homodimer formation and that the extra-terminal domain of nucleoplasm (Fig. 1B). However, although tBRD-2 protein levels tBRD-2 interacts with the C-terminal region of tBRD-1. Peptide were not reduced in homozygous tbrd-1 mutant testes, only a pull-down experiments indicated that tBRD-1 but not tBRD-2 faint tBRD-2 signal was visible in spermatocyte nuclei of preferentially recognizes acetylated histones H3 and H4. Microarray homozygous tbrd-1 mutants (Fig. 1C). By contrast, expression of analyses revealed that several genes are significantly down- a full-length tBRD-1-eGFP fusion protein in the homozygous regulated in tbrd-2-deficient testes. A comparison of different tbrd-1 mutant background reconstituted tBRD-2 localization to microarray data sets demonstrated that tBRD-1, tBRD-2, the tMAC both the chromosomal regions and nucleolus (Fig. 1E′). These component Aly, the Mediator component Med22, and the tTAF Sa results extend our previous analysis and strengthen the idea that share a subset of target genes. Finally, immunofluorescence endogenous tBRD-1 and tBRD-2 interact and that tBRD-2 stainings showed that the sub-cellular localization of tBRD-1 and requires tBRD-1 for proper sub-cellular localization. tBRD-2 requires Aly function. The bromodomains of tBRD-1 are dispensable for homodimer RESULTS formation, and the very C-terminus of tBRD-1 interacts with Expression of tBRD-1-eGFP reconstitutes proper sub-cellular the extra-terminal domain of tBRD-2 localization of tBRD-2 in tbrd-1 mutant spermatocytes Recently, we have shown that tBRD-1 forms homodimers and also Recently, we have shown that the tbrd-1 mutant phenotype is heterodimers with tBRD-2 (Theofel et al., 2014). Here, we aimed rescued by a tbrd-1-eGFP transgene, which contains the tbrd-1 at mapping the interaction domains required for dimerization open reading frame together with 531 bp upstream of the using a series of tBRD-1 and tBRD-2 truncation mutants in the translational start fused in frame with eGFP. The corresponding yeast two-hybrid assay (Fig. 2; Figs S2 and S3). tBRD-1 and tBRD-1-eGFP fusion protein shows the same distribution as tBRD-2 contain several conserved domains, namely the endogenous tBRD-1 (Leser et al., 2012). In addition, we have bromodomains and an extra-terminal domain, which consists of shown that tBRD-1 co-localizes with